Screening culture method for sheep oocytes in vitro

A technique for screening and culturing of oocytes, which is applied in the field of in vitro screening of oocytes, can solve the problem of high blastocyst rate and achieve the effect of high concentration, low concentration and low screening efficiency

Active Publication Date: 2010-06-30
INNER MONGOLIA SAINUO GRASSLAND SHEEP IND
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Problems solved by technology

But the in vitro maturation rate and blastocyst rate of the positive group were higher than those of other groups

Method used

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  • Screening culture method for sheep oocytes in vitro
  • Screening culture method for sheep oocytes in vitro
  • Screening culture method for sheep oocytes in vitro

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Embodiment Construction

[0043] Embodiments of the present invention are described below in conjunction with examples.

[0044] Sheep handling:

[0045] Pick up the sheep ovary within half an hour of slaughter, put it in the normal saline solution containing 1000IU / ml penicillin and 1000IU / ml streptomycin at a temperature of 30℃, wash it three times within 3 hours, and pick out the oocytes under the microscope , add bright cresyl blue at a concentration of 25 μMol / L, place in a 35°C water bath for 80 minutes, and the cytoplasm turns blue for later use.

[0046] Before extracting oocytes, 1.5 ml of oocyte suction liquid must be inhaled for use.

[0047] To screen oocytes, the cytoplasm stained in blue is the positive group (BCB+), and the cytoplasm that is not stained in blue is the negative group (BCB-).

[0048] Preparation of liquid medicine:

[0049] Egg suction solution: TCM199-hepes + 1mg / ml PVA + 0.7mg / ml heparin sodium;

[0050] Bright cresyl blue staining solution: PBS+26μMol / L bright cres...

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Abstract

The invention provides a screening culture method for sheep oocytes in vitro, which comprises the following steps: step 1: ovaries which were killed less than half an hour ago are put into saline water at the temperature of 30DEG C to 40DEG C and containing penicillin and streptomycin, washed for 3 to 4 times within 3 hours, and oocytes are picked out; 25 to 30mu mol / L brilliant cresyl blue is put into 35DEG C to 39DEG C water bath to be dyed for 85 to 92min, the cytoplasm is blue and is washed for 3 to 4 times by in vitro maturation fluid, is put into 55 to 78mul / drip in vitro maturation culture fluid by 25 to 30m / drip, and is cultured in 5 percent CO2 by 95 percent; step 2: cumulus cells are removed from oocytes which are maturated in vitro for 25 to 28 hours; IVF washing liquid is used to wash for 2 to 4 times, and is dripped into 50 to 70mu l fertilization fluid by 25 to 30m / drop; semen is unfrozen, the fertilization fluid is moved in, supernatant fluid is centrifugurated for 4 to 5min and removed, the precipitated sperms are added into fertilization fluid drips by the density of 2 to 4*106 / ml and incubated; eggs which are fertilized for 12 to 18 hours are treated in the above step and then moved into a four-hole culture plate to be cultured; and step 3: reagent egg absorption liquid, brilliant cresyl blue maturation liquid, dyeing liquor SOF, the fertilization fluid, the culture fluid and sheep serum are prepared.

Description

technical field [0001] The invention relates to an in vitro screening technology for oocytes. Brilliant cresyl blue (BCB) staining solution is used to dye and screen sheep oocytes, which can improve the in vitro fertilization rate of oocytes and the in vitro development rate of embryos. Background technique [0002] In vitro screening of oocytes usually adopts the visual evaluation method, which is to classify according to the morphology of the cumulus cell-oocyte complex observed under a microscope and the uniformity of cytoplasm. Grade A: more than three layers of dense cumulus cells with uniform cytoplasm; Grade B: one to three layers of dense cumulus cells with relatively uniform cytoplasm; Grade C: local cumulus cells with uneven cytoplasm; Grade D: naked eggs; E Grade: cumulus cell proliferation. Usually grades A and B are used for mature cultivation, but this method is highly subjective, and there are differences in the judgments of different personnel, and the matur...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/075C12Q1/04A61D19/02
Inventor 黄俊成汪立芹赵云程陈童林嘉鹏
Owner INNER MONGOLIA SAINUO GRASSLAND SHEEP IND
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